Oligosaccharides have several structural forms due to their polyhydroxy character and their terminal carbonyl functions. Both aldoses and ketoses can be present in pyranose, furanose and open chain forms. Furthermore, hemiacetals/hemiketals of oligosaccharides can be characterized by both α and β anomeric linkages. The above described structural diversity makes the isolation of mono and oligosaccharides as a single chemical entity difficult. Selective crystallization of anomeric mixtures of carbohydrates can be rather difficult and quite frequently both anomers are present in crystal structures. Such difficulties often prevent the purification of important carbohydrates via crystallization. The present invention provides a facile approach for the purification of lactosamine hydrochloride via anomer selective crystallization.
Lactosamine hydrochloride is an essential disaccharide present in many natural oligosaccharide structures. To the best knowledge of the present inventors, the single anomeric form of α-lactosamine hydrochloride has never previously been synthesized and characterized. To date, only the anomeric mixture of lactosamine hydrochloride has been prepared, either via chromatography or precipitationt.1-6 The characterization of lactosamine hydrochloride has been rather modest in these publications due to the nature of the anomeric mixture of products. Such lactosamine hydrochloride products have always been described as inseparable anomeric mixtures containing both α and β anomers.
The very first methodology providing anomeric mixtures of lactosamine hydrochloride via crystallization has been described recently.7 The resulting product has been characterized by 1H and 13C NMR. The anomeric mixture nature of the product was indicated by the following characteristic chemical shifts: 1H NMR (D2O) δ=5.23 (d, 1H, H-1α, J1,2=3.1 Hz); δ=4.75 (d, 0.4H, H-1β, J1,2=8.43 Hz); 13C NMR (D2O) δ=103.13 (C-1′ β), 103.08 (C-1′ α), 92.67 (C-1α), 88.99 (C-1β).
An Austrian patent application also describes the precipitation/crystallization approach, also providing an anomeric mixture of lactosamine hydrochloride.8 The described process gave lactosamine hydrochloride as an inseparable mixture containing 60% α- and 40% β-lactosamine hydrochloride proven by the chemical shifts of the two different chemical entities 1H NMR (D2O) (6=5.28 (d, 0.6H, H-1α, J1,2=3.6 Hz); δ=4.81 (d, 0.4H, H-1β, J1,2=8.6 Hz).
The present invention provides crystalline α-lactosamine hydrochloride monohydrate as a single chemical entity, as demonstrated by X-ray crystallography and 1H NMR spectroscopy. Furthermore, the present invention provides anomer selective crystallization methods for lactosamine hydrochloride producing exclusively α-lactosamine hydrochloride monohydrate. No β-lactosamine hydrochloride could be detected in the crystalline product using the most powerful analytical methodologies. The present invention has a great commercial value in large scale production of lactosamine hydrochloride providing high purity of product, which cannot be achieved by any other known purification methods.
Accordingly, the first aspect of the present invention provides α-lactosamine hydrochloride, as shown in Formula 2, substantially free of β-lactosamine hydrochloride.

This product has never previously, to the best knowledge of the present inventors, been prepared and characterized. Preferably, the α-lactosamine hydrochloride comprises less than 5 wt % of β-lactosamine hydrochloride, such as less than 2 wt %, preferably less than 1 wt %, such as 0.5 wt %, and most preferably less than 0.1 wt %.
The present inventors have selectively crystallised the α-form from an anomeric mixture of lactosamine hydrochloride, and have confirmed the identity of the crystalline product to be α-lactosamine hydrochloride monohydrate by means of X-ray crystallography. In addition, the present inventors have prepared anhydrous α-lactosamine hydrochloride.
Anhydrous α-lactosamine hydrochloride having a water content within 0-1% by weight can be made from α-lactosamine hydrochloride monohydrate by removing water of crystallisation via several methods known in the art, such as heating α-lactosamine hydrochloride monohydrate, preferably in vacuum and/or in the presence of a dessicant.
The second aspect of the present invention provides a method of preparation of α-lactosamine hydrochloride monohydrate from an anomeric mixture of lactosamine hydrochloride, comprising:
preparing a solution comprising the anomeric mixture of lactosamine hydrochloride, water and at least one water-miscible organic solvent at a temperature of 0-100° C.;
cooling the solution to cause crystallisation of α-lactosamine hydrochloride monohydrate.
Suitably, the solution is prepared by dissolving the anomeric mixture of lactosamine hydrochloride in water at a temperature of 40-100° C., and subsequently adding the at least one water miscible organic solvent.
Suitably, the method may further comprise the preliminary steps of: dissolving the anomeric mixture of lactosamine hydrochloride in water; and adding the solution to a much larger volume of acetone, such as a tenfold volume of acetone compared with the volume of the aqueous solution, to cause precipitation of a white powder, which powder is then used as the anomeric mixture in preparing the solution comprising the anomeric mixture, water and at least one water miscible organic solvent. This preliminary step forms an acetone adduct of the anomeric mixture that is insoluble in this water/acetone mixture, and so precipitates as a white powder leaving other impurities in solution.
Suitably, the at least one water miscible organic solvent is selected from the group consisting of acetone, tetrahydrofuran, acetonitrile, dioxane, aliphatic alcohols, and 1,2-dimethoxyethane. Suitably, where acetone is used as the at least one water-miscible solvent, the volume of acetone in the solvent mixture is much less than that used to create the acetone adduct, such as up to around 2-3 times the volume of the water in the solvent mixture.
Suitably, the at least one water-miscible solvent is used in greater volume than the volume of water used. Suitably, the water miscible solvent is present at least twice the volume of water used, such as at least 2.5 or 3 times the amount of water used.
Preferably, the at least one water miscible organic solvent is at least one aliphatic alcohol ROH, wherein R is selected from methyl, ethyl, isopropyl, isobutyl and n-butyl. Preferably, the at least one aliphatic alcohol is isopropyl alcohol or isobutyl alcohol. Most preferably, isopropyl alcohol is used.
Preferably, the solution is cooled to between −5 and 30° C. in order to cause crystallisation. Preferably, the solution may be stirred during cooling in order to prevent the formation of large crystals that can capture impurities. This is particularly preferred when crystallising α-lactosamine hydrochloride on a large scale, as the formation of very large crystals in the absence of stirring can cause handling problems and damage to parts of the crystallisation vessel when the crystals are recovered from the vessel. Methods of inducing crystallisation known to the skilled man may also be employed, such as scratching the side of the crystallisation vessel with a glass rod, or providing a seed crystal in the crystallisation vessel. It is particularly preferred to use a seed crystal to induce crystallisation, as this provides better crystal purity and more rapid crystallisation, and the seed crystal used is preferably a crystal of α-lactosamine hydrochloride monohydrate made according to the method of the invention.
The purity of the starting anomeric mixture of lactosamine hydrochloride can vary from 5%-100% of the α-anomer.
The third aspect of the present invention provides a method of preparation of α-lactosamine hydrochloride monohydrate from lactosamine, comprising:
dissolving lactosamine in an aqueous hydrochloric acid solution at 40-100° C.: and
cooling the solution to cause crystallization of the α-lactosamine hydrochloride monohydrate.
Suitably, the concentration of aqueous HCl solution may vary from 0.1% to 38%.
Preferably, the aqueous HCl solution comprises at least one water miscible organic solvent. Suitably, organic solvents such as acetone, tetrahydrofuran, acetonitrile, dioxane, methanol, ethanol, propanol, butanol, or 1,2-dimethoxyethane may be comprised in the aqueous HCl solution. Preferably, the at least one water miscible organic solvent is at least one aliphatic alcohol ROH, wherein R is selected from methyl, ethyl, isopropyl, isobutyl and n-butyl. More preferably, the at least one aliphatic alcohol is isopropyl alcohol or isobutyl alcohol. Most preferably, the at least one aliphatic alcohol is isopropyl alcohol.
Suitably, the solution is cooled to between −5 and 30° C. in order to cause crystallisation. Preferably, the solution may be stirred during cooling in order to prevent the formation of large crystals that can capture impurities. As mentioned above, this is particularly preferred when performing the crystallisation on a large scale. Methods of inducing crystallisation known to the skilled man may also be employed, such as scratching the side of the crystallisation vessel with a glass rod, or providing a seed crystal in the crystallisation vessel. It is particularly preferred to provide a seed crystal to induce crystallisation, as this provides better crystal purity and more rapid crystallisation, and the seed crystal used is preferably a crystal of α-lactosamine hydrochloride monohydrate made according to the method of the invention.
The fourth aspect of the present invention provides a method of preparing anhydrous α-lactosamine hydrochloride having a water content of from 0 to 1% by weight, comprising heating α-lactosamine hydrochloride monohydrate to cause thermal removal of substantially all of the water of crystallisation.
Suitably, the heating temperature can vary from 20-172° C.
Preferably, the method comprises heating the monohydrate under vacuum. Suitably, the method may comprise the use of a dessicant to assist with removal of water of crystallisation.
The present invention further comprises the use of α-lactosamine hydrochloride which is substantially free of β-lactosamine hydrochloride in a food product or beverage, or as a functional food supplement.
The present invention further comprises the use of α-lactosamine hydrochloride which is substantially free of β-lactosamine hydrochloride as an intermediate/precursor for organic synthesis, in particular in the synthesis of human milk oligosaccharides.